We are interested in the transcriptional and post-transcriptional mechanisms of gene regulation during the development of metazoans. The fruit fly Drosophila melanogaster is a good experimental model to address these questions using a combination of genetic and molecular approaches. Two research projects are conducted in the laboratory including (i) the study of the function of the histone-acetyltransferase (HAT) dGcn5 and its role in the genetic and epigenetic control of the development and (ii) the analysis of mechanisms of post-transcriptional silencing by small, double-stranded RNAs.

1. The dGcn5 histone-acetyltransferase

Clément Carré, Caroline Jacquier

Acetylation of histones by multiprotein complexes has been involved in the modulation of chromatin structure and the recruitment of transcription factors to gene promoters. In the lab, we are more particularly interested in understanding how the epigenetic histone code generated by histone-acetyltransferases (HAT) contributes to genetic programs during development.

We conducted an extensive EMS-induced mutagenesis, and isolated and characterized four loss-of-function mutations of the DrosophilaGcn5 gene. The loss of function of Gcn5 blocks the onset of both oogenesis and metamorphosis and hypomorphic Gcn5 alleles impair the ecdysone-controlled formation of adult appendages and cuticle. Strikingly, Gcn5 is not required for larval development. In contrast there are strong cell proliferation defects in Gcn5-depleted imaginal tissues. In vivo global histone H3 acetylation of K9 and K14 lysine residues was lost in Gcn5 mutants, while other histone modifications were not affected (Fig. 1). Together, these results pointed out to Gcn5 as the major histone H3 acetylase in Drosophila involved in the control of specific morphogenetic cascades during development.

The Drosophila Gcn5 protein is the catalytic unit of at least two multiprotein complexes of distinct composition, ATAC and dSAGA. Using Affymetrix® microarrays, we analyzed over the past year the transcriptome changes induced by the loss of function of dGcn5. We collaborate in a consortium of European labs that are performing the same kind of microarray analyses with various mutants for the components of the ATAC and dSAGA complexes. The microarray data are currently clustered and compared and should provide essential information to assign specific functions to both of these complexes.

We have also shown that histone H3 acetylation by dGcn5 is impaired in mutant background for another HAT, the dCBP protein. We are currently analyzing the molecular mechanisms of the functional interaction between dGcn5 and dCBP. Last, we observed that the X chromosome is dramatically decondensed in dGcn5 male mutants, a defect that is also observed in mutants for components of the NURF ATPase-dependent remodeling complex. We are currently testing the genetic interactions between these components and dGcn5 and we are investigating the contribution of the dGcn5-dependent histone H3 acetylation to the targeting to the NURF complex to the chromatin.

2. Analysis of Genetic Interference by small, double-stranded RNAs.

Hélène Thomassin, Delphine Fagegaltier et Bassam Berry.

RNA interference (RNAi) designates the process by which double-stranded RNA induce the specific degradation of their complementary mRNA. This process is involved in various regulatory pathways in eukaryotes, including defense against plant viruses, heterochromatinization of pericentromeric regions, various genetic cosuppression phenomena, repression of transposable elements and chromosome imprinting.

Micro-RNAs are short hairpin, partially double stranded RNA encoded by eukaryote genomes. Several hundreds of micro-RNA have been recently described whose maturation and mode of action involves pathways overlapping with RNAi pathways. However, instead of functioning as guide for the degradation of their target mRNA, micro-RNA inhibit their translation. Although only recently characterized, it is clear that micro-RNA are involved in the regulation of essential processes such as development, cell proliferation and apoptosis.

We developed a method to trigger RNAi in vivo using double-stranded RNA producing transgenes (Fig. 2). This method turned out to be a powerful approach to inactivate drosophila genes in a tissue- or stage-specific manner. It was used to inactivate the EcR gene, which encodes the drosophila nuclear receptor of the steroid hormone ecdysone. In collaboration with the Jim Truman lab, this approach allowed to demonstrate the role of the EcR nuclear receptor in the differentiation of wing sensory bristles. On the other hand, the RNAi induced EcR inactivation was used in collaboration with the Pierre Leopold lab to demonstrate that the steroid hormone ecdysone is involved in a negative feedback loop antagonizing insulin-dependent cell growth.

Two projects are currently under development to study the contribution of small, double-stranded RNA to the endogenous regulatory mechanisms of gene expression in Drosophila.

We have built, in collaboration with Pasteur-Génopole®, LNA oligonucleotide microarrays to perform genome wide analyses of micro-RNA expression profiles. Using this new tool, we are going to search for miRNAs specifically induced at the onset of metamorphosis and potentially involved in the hormonally regulated genetic cascade that orchestrate development during this period. We are also interested in characterizing miRNAs involved in sexual differentiation, aging and responses to oxidative stresses.

Another experimental approach consists in perturbing small RNA pathways by expressing in transgenic flies viral suppressors of RNA silencing from plant and insect viruses. We are currently testing the effects of several suppressors on RNAi and miRNA pathways as well as on chromatin structure modulation. We are also testing whether these suppressors increase the susceptibility of transgenic animals to a viral infection.

Legend to Photos :

Figure 1: Left hand side. Spreading of polytene chromosomes from salivary glands of wild type or Gcn5 mutant third instar larvae, as indicated. Immunostaining using a specific antibody (red) revealed that acetylation of H3-K14 residue is lost in Gcn5 mutants.

Right hand side. An RNAi transgenic system (see below) was used to silence Gcn5 in the posterior compartment of imaginal discs. Acetylation of H3-K14 residue was lost in that compartment.

Figure 2: A transgenic system to target RNAi in Drosophila

Left hand side. A transgenic line expressing GAL4 under the control of a specific driver is crossed with a transgenic line transgenic for an inverted repeat under the control of UAS regulatory sites.